CENTRAL ANALGESIC ACTIVITY OF Litsea polyantha Juss. BARK EXTRACT - PowerPoint PPT Presentation

CENTRAL ANALGESIC ACTIVITY OF Litsea polyantha Juss. BARK EXTRACT Manik Ghosh * and Barij Nayan Sinha Department of Pharmaceutical Sciences and Technology Birla Institute of Technology, Mesra, Ranchi, Jharkhand (835215), INDIA *Corresponding Author’s E-mail: manik@bitmesra.ac.in Tel.: + 916512276247; Fax: + 916512275290

ABSTRACT

The sensation of pain is initiated in peripheral pain receptors (nociceptors) and its purpose is to draw attention to tissue damage. In order to test analgesic activity, it is obviously necessary to induce pain in the subject and then modify the response to, or perception of, this pain. Analgesic studies of the methanol (90% v/v) extract (MELP) of Litsea polyantha Juss. bark (Yield: 11.79% w/w) was carried out using healthy adult Swiss albino mice of either sex weighing between 20 to 25 g respectively. The experiment protocols were approved by the Institutional Animal Ethical Committee (621/02/ac/CPCSEA) prior to the conduct of the animal experiments. The animals were divided into 6 groups (n=6). Group I and II were used as control, received 10% v/v propylene glycol (PG) and distilled water (DW) at the dose of 10 ml/kg b.w. Group III, IV & V were treated with MELP (50, 75 and 100 mg/kg b.w., i.p.), respectively; Group VI received Morphine sulphate (10 mg/kg b.w., s.c.) an opioid analgesic as standard drug. A reduction in the tail withdrawal as compared to the control group was considered as evidence for the presence of analgesia. Tail flick latency was measured 30 min after the drug administration and Pain Inhibition Percentage (PIP) was calculated. MELP given by intraperitoneal route in mice showed significant and dose-dependent central analgesic activity (P<0.001) at all dose levels. MELP showed 22.2% – 60.4% increase in PIP in tail flick test and 21.2% – 67.8% increase in PIP in tail immersion method. 3

INTRODUCTION

Natural Products • Natural products have been the single most productive source of leads for the development of drugs. • Over a 100 new products are in clinical development, particularly as anti-cancer agents and antiinfectives. • Comparisons of the information presented on sources of new drugs from 1981 to 2007 indicate that almost half of the drugs approved since 1994 are based on natural products. Harvey AL. Natural products in drug discovery. Drug Discov. Today 2008; 13(19/20): 894-901

Natural Product-Derived Drugs at Different Stages of Development Pre- Phase Phase Phase Pre- Therapeutic area registration Total clinical I II III Cancer 34 15 26 9 2 86 Anti-infective 25 4 7 2 2 40 Neuropharmacological 6 3 9 4 0 22 Cardiovascular / 9 0 5 6 0 20 gastrointestinal Inflammation 6 2 9 1 0 18 Metabolic 7 3 6 1 0 17 Skin 7 1 2 0 0 10 Hormonal 3 0 2 1 0 6 Immunosuppressant 2 2 0 2 0 6 Total 99 30 66 26 4 225 Harvey AL. Natural products in drug discovery. Drug Discov. Today 2008; 13(19/20): 894-901

Folklore Medicines • Large numbers of medicinal plants have been advocated in folklore medicines of Jharkhand for treating various diseases and disorders. • One of such a plant is Litsea polyantha Juss., locally known as Pojo. • Tribal of Chotanagpur region are using bark of this plant for treatment of different diseases and ailments like pains, inflammation, bruises & contusions, cuts, wounds, diarrhea and fractures in animals. 7

Litsea • Litsea, a large genus comprising of around 700 species of evergreen trees or shrubs, distributed chiefly in tropical and subtropical Asia, Australia and the Pacific Islands. About 43 species are found in India. It belongs to the family Lauraceae. The Wealth of India (CSIR), 1985; (VI):152-156 http://www.tropicos.org/TaxonomyBrowser.aspx?nameid=40007934&conceptid=1 8

Ethnopharmacology • Parts used : – In Folklore medicines - Bark, Stem and Roots are used to treatment various diseases and disorders . • Properties and uses – The bark of Litsea polyantha Juss. is mildly astringent and is reported to be used for diarrhea. – Powdered bark and roots are used for treatment of cuts, wounds, pains, bruises and contusions. – The Powdered bark is also used to cure fractures in animals. – The seed fat is use in ointments for rheumatism. 1. The Wealth of India (CSIR), 1985; (VI):154-155 2. Kirtikar KR and Basu BD. Indian Medicinal Plants, M/S periodical Experts, Delhi, 2nd ed., 1935 (Reprint 1975); (III): 2160-2161 9

EXPERIMENTAL 10

Collection and Authentication • The bark of Litsea polyantha Juss (Lauraceae) were collected from BIT, Mesra of Ranchi District. • The parts were authenticated by Dr. S. Jha, Professor, Department of Pharm. Sciences, BIT, Mesra and Dr. P. Venu, Scientist ‘F’ & HOO, Botanical Survey of India, Central National Herbarium, Howrah. • The voucher specimen (BIT 417) was preserved in the Department of Pharmaceutical Sciences, BIT, Mesra. 11

Drying and Size Reduction • The bark of Litsea polyantha Juss were dried in shade for about a week followed by drying at 35 ° C – 40 ° C in oven for 1 day. The dried barks were then grinded to coarse powder in an iron mortar and pestle. This powdered material was again dried in the oven at 35 ° C . 40 ° C for 1 hour and used for extraction. Extraction • The dried and powdered plant material (Bark) was subjected to successive hot extraction in a soxhlet apparatus with solvents of increasing polarity viz. petroleum ether (60-80), chloroform, ethyl acetate and methanol. • The average time period for extraction was 48 hours. The extract was then filtered using Whatman No. 1 filter paper and the filtrate was distilled followed by evaporation in a vacuum rotary evaporator. Methanol extract was also subjected to lyophilization. 12

• In another extraction method the defatted bark of Litsea polyantha Juss. was subjected to hot extraction in a soxhlet apparatus with mixture of methanol and water (90:10). • The average time period for extraction was 72 hours. The extract was then filtered using Whatman filter paper No. 1, the filtrate was distilled and evaporated in a vacuum rotary evaporator followed by lyophilization. 13

Extractive Values and color of methanol (90% v/v) extract of Litsea polyantha Juss. bark % Yield Name of the Color of Color at Color at (Hot) Consistency Extract Extract 365 nm 254 nm w/w Petroleum ether 1.03 Pale White White Yellow Greasy Waxy (60-80) – PLP Methanol Reddish 11.79 Black Light blue Amorphous (90 % v/v) – MELP Brown 14

PHARMACOLOGICAL INVESTIGATIONS

SELECTION OF EXTRACT FOR PHARMACOLOGICAL INVESTIGATIONS • All the five extracts were concentrated in rotary evaporator followed by lyophilization as and when required. The completely dried samples were then reconstituted with 10% v/v propylene glycol (PG) for pharmacological experiments. • Guided by the ethnopharmacological literatures on Litsea polyantha Juss., all the five extracts were subjected to pharmacological screening. Results suggested that methanol (90% v/v) extract (MELP) was pharmacologically more potent than other extracts. • The percentage yield of methanol (90% v/v) extract (MELP) was also appreciably high (11.79 % w/w). This extract answered positive for major phytoconstituents like alkaloids, flavonoids, etc present in Litsea species. This is how MELP was selected for detail pharmacological and phytochemical investigations. 16

Central Analgesic Activity • Tail Flick Method • Tail Immersion Method •Eddy’s Hot Plate Method

Tail Flick Method Effect of MELP on Tail flick response in Swiss albino mice. Response Time (sec) Mean ± SEM (n=6) Time (min) PG DW MELP 50 MELP 75 MELP 100 Morphine 7.12 ± 6.87 ± 7.10 ± 7.06 ± 6.91 ± 6.78 ± 0 0.09 0.13 0.20 0.17 0.14 0.15 6.90 ± 7.14 ± 8.63 ± 10.03 ± 11.06 ± 11.90 ± 30 0.16 0.20 0.16* 0.26* 0.28* 0.38* -2.91 ± 3.93 ± 22.21 ± 42.68 ± 60.42 ± 76.28 ± PIP 3.38 2.33 5.09 5.96 4.77 5.12 Values reported as Mean ± SEM (n=6). The data were analyzed by two way ANOVA followed by Bonferroni's Multiple Comparison Test. Asterisk indicated statistically significant values from control. * P<0.001 . PG: Propylene Glycol; DW: Distilled Water; MELP: Methanol (90% v/v) extract of Litsea polyantha Juss. bark; PIP: Pain Inhibition Percentage. Turner, R.A., 1965. In: Turner, R., Ebborn, P. (Eds.), Analgesics: Screening Methods in Pharmacology. Academic Press, New York. 18

Tail Flick Method Effect of MELP on tail flick response in Swiss albino mice. Values reported as Mean ± SEM (n=6). The data were analyzed by two way ANOVA followed by Bonferroni's Multiple Comparison Test. Asterisk indicated statistically significant values from control. * P<0.001 . PG: Propylene Glycol; DW: Distilled Water; MELP: Methanol (90% v/v) extract of Litsea polyantha Juss. bark; PIP: Pain Inhibition Percentage. 19